Relation of patient and pathology characteristics with the Ins/Cho ratio
The mean and median values of the Ins/Cho ratios for the 30 patients included in this cohort were 0.75 and 0.67, respectively. We opted for 0.7 as the cutoff value and classified those patients into high (≥0.7) and low (<0.7) Ins/Cho ratio groups (n = 13 and 17, respectively). Tables 1 and 2 show the patient characteristics, pathologies, Ins/Cho ratios, and postoperative therapies. Case comprised 25 glioblastoma, two gliosarcomas, three astrocytomas. Two of three astrocytomas gained chromosome 7 and/or loss of chromosome 10q, but the other was not associated with them. There were no statistically significant differences in sex, age at onset, laterality, MIB-1 index, or grade Ⅳ WHO classification between the two groups. Overall, eight patients underwent reoperation, six patients underwent additional chemotherapy, and seven patients had no treatment after recurrence.
Table 1. Characteristics and treatments of patients
Case
|
Age, sex
|
Pathology
|
Recurrence
|
PFS
|
Follow-up terms
|
Outcome
|
Ins/Cho
|
First operation / Postoperative therapies / therapies after recurrence
|
1
|
42F
|
GBM, Gr Ⅳ
|
Yes
|
9
|
22
|
Dead
|
0.573
|
GTR / TMZ + RT / TMZ + RT + BEV+ other chemotherapies + operation
|
2
|
43M
|
GBM, Gr Ⅳ
|
Yes
|
5
|
12
|
Dead
|
0.475
|
PR / TMZ + RT / BEV + another chemotherapy + operation
|
3
|
43F
|
GBM, Gr Ⅳ
|
Yes
|
1
|
31
|
Dead
|
0.507
|
GTR / No treatment / TMZ + RT + BEV + other chemotherapies + operations
|
4
|
44M
|
GBM, Gr Ⅳ
|
Yes
|
10
|
14
|
Dead
|
0.167
|
GTR / TMZ + RT + BEV / other chemotherapy
|
5
|
46F
|
GBM, Gr Ⅳ
|
Yes
|
3
|
12
|
Dead
|
0.450
|
GTR / TMZ + RT / TMZ + RT + BEV + other chemotherapy
|
6
|
62M
|
GBM, Gr Ⅳ
|
Yes
|
14
|
49
|
Dead
|
0.516
|
Biopsy / TMZ + RT / TMZ + RT + BEV + operation
|
7
|
62M
|
GS, Gr Ⅳ
|
Yes
|
26
|
58
|
Alive
|
1.149
|
STR / TMZ + RT / TMZ + RT + operation
|
8
|
63F
|
GBM, Gr Ⅳ
|
Yes
|
17
|
36
|
Alive
|
1.149
|
GTR / TMZ + RT / TMZ + RT + BEV
|
9
|
67M
|
GBM, Gr Ⅳ
|
Yes
|
2
|
2
|
Alive
|
0.044
|
PR / TMZ + RT / BEV
|
10
|
67M
|
GBM, Gr Ⅳ
|
Yes
|
9
|
10
|
Alive
|
0.816
|
PR / TMZ + RT + BEV / TMZ
|
11
|
68F
|
GBM, Gr Ⅳ
|
Yes
|
8
|
8
|
Dead
|
0.227
|
GTR / TMZ + RT / No treatment
|
12
|
68M
|
GBM, Gr Ⅳ
|
NO
|
6
|
10
|
Alive
|
1.182
|
STR / TMZ + RT / No treatment
|
13
|
69F
|
GBM, Gr Ⅳ
|
Yes
|
1
|
12
|
Dead
|
2.054
|
GTR / No treatment / TMZ + RT + BEV
|
14
|
69F
|
GBM, Gr Ⅳ
|
Yes
|
12
|
20
|
Dead
|
0.655
|
GTR / TMZ + RT / BEV
|
15
|
70M
|
GBM, Gr Ⅳ
|
NO
|
3
|
11
|
Dead
|
0.854
|
GTR / TMZ + RT / No treatment
|
16
|
71M
|
DA, Gr Ⅱ
|
Yes
|
9
|
39
|
Dead
|
1.702
|
Biopsy / TMZ + RT / operation
|
17
|
72M
|
GBM, Gr Ⅳ
|
Yes
|
1
|
22
|
Dead
|
0.451
|
GTR / TMZ + RT / TMZ + RT + BEV
|
18
|
72F
|
DA, Gr Ⅱ
|
Yes
|
4
|
7
|
Alive
|
0.952
|
PR / No treatment / No treatment
|
19
|
73F
|
GBM, Gr Ⅳ
|
Yes
|
1
|
23
|
Dead
|
0.405
|
STR / No treatment / TMZ + RT + BEV + operation
|
20
|
74M
|
GBM, Gr Ⅳ
|
Yes
|
2
|
20
|
Dead
|
0.758
|
GTR / TMZ + RT / TMZ + BEV
|
21
|
74M
|
GBM, Gr Ⅳ
|
Yes
|
1
|
20
|
Dead
|
0.240
|
GTR / TMZ + RT / TMZ + BEV + another chemotherapy + Operation
|
22
|
74M
|
GBM, Gr Ⅳ
|
Yes
|
10
|
19
|
Alive
|
0.893
|
STR / TMZ + RT + BEV / TMZ + RT + BEV
|
23
|
75F
|
GBM, Gr Ⅳ
|
NO
|
6
|
6
|
Alive
|
0.405
|
GTR / TMZ + RT
|
24
|
76F
|
AA, Gr Ⅲ
|
Yes
|
1
|
24
|
Dead
|
0.635
|
Biopsy / TMZ + RT / TMZ + BEV
|
25
|
77M
|
GS, Gr Ⅳ
|
Yes
|
13
|
14
|
Dead
|
0.695
|
GTR / TMZ + BEV / No treatment
|
26
|
80F
|
GBM, Gr Ⅳ
|
NO
|
39
|
39
|
Alive
|
1.176
|
PR / TMZ + RT
|
27
|
81M
|
GBM, Gr Ⅳ
|
Yes
|
2
|
3
|
Dead
|
0.692
|
Biopsy / TMZ + BEV / No treatment
|
28
|
81M
|
GBM, Gr Ⅳ
|
Yes
|
4
|
4
|
Alive
|
1.397
|
GTR / No treatment
|
29
|
85M
|
GBM, Gr Ⅳ
|
Yes
|
2
|
5
|
Alive
|
0.279
|
Biopsy / TMZ + BEV +RT / TMZ
|
30
|
86F
|
GBM, Gr Ⅳ
|
Yes
|
21
|
24
|
Dead
|
1.111
|
STR / TMZ / BEV
|
Data show the patient and treatment details after the first operation and after recurrence for supratentorial IDH wild-type glioma (n=30). Pathology was diagnosed according to the WHO classification 2016. Abbreviations: AA, anaplastic astrocytoma; BEV, bevacizumab; DA, diffuse astrocytoma; F, female; GBM, glioblastoma; Gr, grade; GS, gliosarcoma; GTR, Gross total resection; Ins/Cho, ratio of myoinositol to total choline; IDH, isocitrate dehydrogenase; M, male; PFS, progression free survival; PR, Partial resection; RT, radiotherapy; STR, Subtotal resection; TMZ, temozolomide.
Table 2. Comparison of patient characteristics by Ins/Cho ratio threshold
|
Ins/Cho ratio≥0.7
(n = 13)
|
Ins/Cho ratio<0.7
(n = 17)
|
P value
|
Male, n
|
8
|
9
|
0.72
|
Age at onset, median (years)
|
71
|
69
|
0.38
|
Left side, n
|
8
|
7
|
0.46
|
MIB-1 index, median
|
38
|
46
|
0.41
|
Grade Ⅳ, n
|
11
|
16
|
0.57
|
Abbreviations: Grade Ⅳ, grade Ⅳ in WHO Classification; Ins/Cho, ratio of myoinositol to total choline; n, number of patients.
PFS and OS analysis
We compared prognosis by the Ins/Cho ratio. During follow-up, tumor recurrence occurred in 94% of patients with low Ins/Cho ratio (i.e., 16/17) and in 77% with high Ins/Cho ratio (i.e., 10/13). Moreover, 82% (14/17) of patients with low ratios and 38% (5/13) with high ratios died. Figures 1a and 1b show that the PFS and OS in those with high ratios were significantly shorter than in those with low ratios, indicating higher recurrence and mortality rates in the patients with low Ins/Cho ratios (P = 0.020 and 0.037, respectively). In the multivariate analysis, Cox proportional hazards models revealed that the Ins/Cho ratio was significantly associated with PFS (hazard ratio 0.34, P = 0.027), which suggested that the Ins/Cho ratio was useful outcome predictor, especially for PFS. These data are summarized in Tables 3a and 3b.
Table 3a. Multivariate analysis of PFS
Explanatory factor
|
Hazard ratio (95% CI)
|
P value
|
GTR or STR
|
0.84 (0.36-2.0)
|
0.68
|
Use of BEV
|
0.76 (0.26-2.2)
|
0.62
|
Both use of TMZ and RT
|
0.52 (0.20-1.4)
|
0.18
|
Ins/Cho ratio
|
0.34 (0.13-0.88)
|
0.027
|
Table 3b. Multivariate analysis of OS
Explanatory factor
|
Hazard ratio (95% CI)
|
P value
|
GTR or STR
|
1.6 (0.44-5.6)
|
0.48
|
Use of BEV
|
0.91 (0.28-3.0)
|
0.88
|
Both use of TMZ and RT
|
0.70 (0.22-2.2)
|
0.55
|
Ins/Cho ratio
|
0.33 (0.10-1.1)
|
0.067
|
Tables 3a and 3b show the results of multivariate analysis: we chose GTR or STR, BEV, TMZ and RT, and the Ins/Cho ratio as explanatory factors and analyzed their relevance to PFS and OS. Abbreviations: BEV, bevacizumab; GTR, Gross total resection; Ins/Cho, ratio of myoinositol to total choline; OS, overall survival; PFS, progression free survival; RT, radiotherapy; STR, subtotal resection; TMZ, temozolomide.
Analysis of copy number aberration by next generation sequencing
We included 20 cases in the next generation sequencing, analyzing nine samples in the group with high Ins/Cho ratios (≥0.7) and 11 samples in the group with low Ins/Cho ratios (<0.7). The overall noise value was <0.3. Analysis focused on regions where specific genes exist that are associated with poor prognosis in glioblastoma, including 7p11.2 (EGFR), 9p21.3 (p16), 10q23.3 (PTEN) [15, 16]. Gain of 7p11.2 was detected in seven cases with high ratios and in six cases with low ratios, loss of 9p21.3 was detected in four cases with high ratios and in three cases with low ratios, and loss of 10q23.3 was detected in four cases with high ratios and in three cases with low ratios (Table 4). There were no significant differences between the groups in any other window.
Table 4. Comparison of copy number aberrations by Ins/Cho ratio
|
Ins/Cho ratio ≥0.7
(n = 9)
|
Ins/Cho ratio <0.7
(n = 11)
|
P value
|
+7p11.2
|
7 (78%)
|
6 (55%)
|
0.37
|
−9p21.3
|
4 (44%)
|
3 (27%)
|
0.64
|
−10q23.3
|
4 (44%)
|
3 (27%)
|
0.64
|
Copy number aberrations identified by next generation sequencing are compared by the Ins/Cho ratio. We defined an effective change of gain or loss when the average value of a window was >2.5 or <1.5. Abbreviations: Ins/Cho, ratio of myoinositol to total choline.